The cross-regulation of Gi-protein by cholera toxin involves a phosphorylation by protein kinase A.

Pretreatment of alveolar macrophages with cholera toxin inhibits the release of arachidonic acid induced by the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. The results presented here show that cholera toxin might exert its inhibitory effect through the phosphorylation of Gi alpha by protein kinase A (PKA). (1) Gi-proteins from cells pretreated with cholera toxin showed parallel increases in their sensitivity to ADP-ribosylation by toxins in vitro and in Gi alpha phosphorylation. By contrast, the Gi alpha concentration was unchanged. (2) Cholera toxin pretreatment also decreased the functional activity of Gi, as assessed by the inhibition (80%) of agonist-induced binding of guanosine-5'-[gamma-thio]triphosphate (GTP[gamma S]). (3) These effects of cholera toxin were blocked by a specific PKA inhibitor, N-(2-[methyl-amino]ethyl)-3-isoquinolinesulphonamide dihydrochloride (H8) and mimicked by a cyclic AMP (cAMP) analogue and a phosphatase inhibitor. (4) Gi alpha was also phosphorylated in vitro by the catalytic subunit of PKA. In contrast with other cell systems, the stimulation of protein kinase C seems to have no effect on the sensitivity of Gi to ADP-ribosylation or on its phosphorylation. Therefore, the phosphorylation of Gi-proteins by PKA seems to be the actual target of the negative control of arachidonic acid release via the cAMP-mediated pathway.

Increase of bradykinin-stimulated arachidonic acid release in a delta F508 cystic fibrosis epithelial cell line.

Modification of chloride conductance by bradykinin in epithelial cells has been attributed to an activation of protein kinase A resulting from adenylcyclase stimulation by arachidonic acid cyclooxygenase products. The results presented here compare tracheal epithelial cell lines from one control and two cystic fibrosis patients which were immortalized by transfection with the SV40 large T oncogene. The three cell lines presented the same arachidonic acid content, turnover and mobilisation under basal conditions. Bradykinin stimulated the release of arachidonic acid and the synthesis of cyclooxygenase derivatives (mainly PGE2). The cell line from the cystic fibrosis patient bearing a phenylalanine 508 deletion, which is the major form of the disease, showed a higher bradykinin-induced arachidonic acid release than either control cells or cells from a patient presenting a minor form of the disease. This higher sensitivity suggests a dysregulation of phospholipase A2 stimulation in cystic fibrosis cells and was confirmed on non-immortalized tracheal epithelial cells in primary culture and on skin fibroblasts from patients bearing the same mutation. This defect is associated with a potentiation of cholera toxin pretreatment on cAMP content of delta F508 cell line. The impaired control of arachidonic acid release cannot be attributed to an increased number of bradykinin binding sites, since this increase was similar in the two cystic fibrosis cell lines.

Stimulatory and inhibitory guanine-nucleotide-binding regulatory protein involvement in stimulation of arachidonic-acid release by N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor from guinea-pig alveolar macrophages. Differential receptor/G-protein interaction assessed by pertussis and cholera toxins.

The involvement of guanine-nucleotide-binding regulatory proteins (G proteins) in the regulation of arachidonic-acid release induced by N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or platelet-activating factor (PAF) was examined in guinea-pig alveolar macrophages. We report that maximal release of arachidonic acid in permeabilized cells requires the simultaneous addition of the agonist (fMet-Leu-Phe or PAF) and of GTP (or GTP[S]). Prior treatment of cells with increasing concentrations of pertussis toxin induces a parallel decrease of arachidonic-acid release and of the labeling of a 40-kDa protein in membranes incubated with [32P]NAD and pertussis toxin. fMet-Leu-Phe, but not PAF, allows the ADP-ribosylation of a 40-KDa protein by cholera toxin in the presence of Mg2+. This effect is prevented by guanyl nucleotides and by prior treatment with pertussis toxin. The 40-kDa protein ADP-ribosylated seems to be alpha i1 and/or alpha i2. Stimulation of GTPase activity by fMet-Leu-Phe and PAF has the same amplitude and is completely inhibited by pertussis toxin, but only in part by cholera toxin. Prior treatment of alveolar macrophages with cholera toxin, which ADP-ribosylates Gs, inhibits PAF-stimulated and fMet-Leu-Phe-stimulated arachidonic-acid release to the same extent, via a cAMP-protein-kinase-A cascade. The decreased responsiveness of alveolar macrophages previously treated with cholera toxin to fMet-Leu-Phe and PAF is associated with a strong increase of in-vitro [32P]NAD labeling of Gi proteins either by pertussis or by cholera toxin. This effect is mimicked by prior treatment of the cells with dibutyryl cAMP and okadaic acid, a protein-phosphatase inhibitor, suggesting the involvement of protein-kinase A in this process. In conclusion, our results demonstrate that fMet-Leu-Phe and PAF receptors interact differently with Gi1/2 proteins in guinea-pig alveolar macrophages. Gi1/2 proteins are a possible target of the cross-regulation of arachidonic-acid release by a Gs-mediated pathway.